Enzyme and Microbial Technology 36 (2005) 520–526

Enzymatic production ofd-p-hydroxyphenylglycine fromdl-5-p-hydroxyphenylhydantoin bySinorhizobium morelensS-5

Sheng Wu, Liu Yang, Yanbin Liu, Guogang Zhao, Jianjun Wang, Wanru Sun∗

State Key Laboratory of Microbial Resources, Institute of Microbiology, Chinese Academy of Sciences, 100080 Beijing, PR China

Received 18 August 2004; accepted 18 November 2004

Abstract

A microorganism with the ability to formd-p-hydroxyphenylglycine (d-pHPG) fromdl-5-p-hydroxyphenylhydantoin (dl-5-pHPH) wasisolated and identified asSinorhizobium morelensS-5. Hydantoinase and carbamoylase involved in this bioconversion process were bothstrictly d-stereospecific. Addition ofdl-5-(2-indolymethyl)hydantoin in the medium could enhance the biotransfromation ability of the cellsof S. morelensS-5. The optimum temperature and pH ford-pHPG production, respectively, were 45◦C and 8.2 when resting cells were usedduring the biotransformation process. Partially purifiedd-carbamoylase exhibited a temperature optimum at 60◦C and pH optimum at 7.0 in0©

KH

1

ts[ta5atpddtpvpg

rentneous.

e-

ism,e--

llsellspar-

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.1 M phosphate buffer. The thermostablility of the enzyme was remarkable, with no loss of activity detected after 40 min at 50◦C.2004 Elsevier Inc. All rights reserved.

eywords: Sinorhizobium morelens; d-Hydantoinase;d-Carbamoylase;d-p-Hydroxyphenylglycine;N-Carbamoyl-d-p-hydroxyphenylglycine;dl-5-p-ydroxyphenylhydantoin; Stereoselectivity

. Introduction

Optically actived-amino acids are widely used as in-ermediates in the pharmaceutic field for the production ofemisynthetic antibiotics, peptides, hormones and pesticides1]. Among them,d-pHPG is used as a side chain for semisyn-hetic penicillins and cephalosporins such as ampicillin andmoxicillin. Chemical hydrolysis of 5-pHPH gives racemic-pHPG, which requires resolution to optically pure aminocids. This procedure makes the process cumbersome. Con-

rary to this, enzymatic production ofd-pHPG fromdl-5-HPH became increasingly attractive. In the process, the-5-pHPH is selectively hydrolyzed to formN-carbamoyl-pHPG byd-sterospecific hydantoinase (E.C.3.5.2.2), and

hen the later is further hydrolyzed to remove carbamoyl androduced-pHPG by a carbamoylase[2–4]. The major ad-antage of this reaction system is that the racemization of 5-HPH spontaneously carried out under alkaline conditions,iving a theoretical yield of 100% rather than 50%.

∗ Corresponding author. Tel.: +86 10 62587206; fax: +86 10 62653468.

Microbial hydantoinase and carbamoylase have diffesubstrate- and stereospecifities and are very heterogeAt present, only a few microorganisms[2,4–7]are known tohave the ability of producingd-amino acids from the corrsponding 5-substituted hydantoins.

This paper reports the isolation of a novel microorganSinorhizobium morelensS-5, showing the ability to sterospecifically convertdl-5-pHPH tod-pHPG in one-step process. Improvement of thed-pHPG production by resting cewas achieved by optimizing the cultural conditions of cgrowth and bioconversion conditions. The properties oftially purified d-carbamoylase are also reported.

2. Materials and methods

2.1. Chemicals

dl-5-Monosubstituted hydantoins were prepared fromcorrespondingdl-amino acids according to the procedureSuzuki et al.[8]. N-carbamoyl-d-pHPG was synthesized

E-mail address:sunwr@sun.im.ac.cn (W. Sun). the method of Stark and Smyth[9]. dl-pHPG was prepared

141-0229/$ – see front matter © 2004 Elsevier Inc. All rights reserved.oi:10.1016/j.enzmictec.2004.11.007

S. Wu et al. / Enzyme and Microbial Technology 36 (2005) 520–526 521

by racemizing thed-pHPG in a strong alkaline solution. sul-fonyl �-cyclodextrin (degree of substitution (DS) 13) waspurchased from Acros Company. Triethanolamine was pur-chased from Merck Company. All other chemicals used inthese experiments were of analytical grade.

2.2. Screening methods

The isolation of the microorganisms has been performedon medium I. Liquid samples from soil were spread on agarPetri-dishes of the above medium. Once colonies appeared,each one was grown in the same liquid medium, and then re-isolated on the agar medium until a pure colony was obtained.The microorganism isolated for its capacity to hydrolyzedl-5-pHPH orN-carbamoyl-d-pHPG tod-pHPG was confirmedusing biocatalytic assays of resting cells.

2.3. Microorganisms and cultivation media

A Gram-negative bacterium,S. morelensS-5 isolatedfrom soil, was used in all experiments. Medium I used forthe screening experiments contained per liter: 10.0 g glu-cose, 3.0 g NaCl and 1.0 gN-carbamoyl-d-pHPG at pH 7.2.Medium II contained per liter: 10.0 g glucose, 10.0 g yeastextract, 3.0 g NaCl and 1.0 gN-carbamoyl-d-pHPG at pH7 ter-m yn-t d in-d tiono .0 gg0i

2

f from1t ndr .0)c asc n-t ana-l eachtd ram( berop

2

c nt ged

(10,000×g, 10 min) and the supernatant diluted with deion-ized water. Analyses were performed by HPLC, usinga C18 column (DiamosilTM C18, Dikman TechnologiesCo., China); column size: 4.6 mm× 250 mm; mobile phase:H2O/CH3CN/85% H3PO4 (95/5/0.01 by volume); flow rate:1 ml/min; and detector: UV detector at 230 nm. Retentiontimes:d-pHPG, 2.7 min;N-carbamoyl-d-pHPG, 8.1 min;dl-5-pHPH, 11.6 min. Biomass concentration was estimated byoptical density (OD) measurement at 600 nm. OD was cali-brated against cell dry weight (CDW).

2.6. Products isolation and identification

The bioconversion broth was centrifuged and the super-natant was applied to preparative HPLC (system above). Thecomponents were collected and freeze–dried, then recrystal-lized in ethanol/H2O. Identification ofN-carbamoyl-d-pHPGandd-pHPG was achieved by comparison of infrared (IR) and1H-nuclear magnetic resonance (NMR) spectra with those ofthe authentic products. The enantiomeric excess was deter-mined by chiral capillary electrophoresis (CE). CE was car-ried out with using a P/ACE MDQ capillary electrophoresisinstrument (Beckman Coulter Co.) equipped with an on-lineUV detector. CE was performed by using a fused silica cap-illary tube (length 37 cm, effective length 30 cm, i.d.75�m);T OHf n-n ul-f pH2 lvedi allyi tubet aso de-t

2

wasm ona er-s moy-l ffer,p ni-c lari-fi ntw r-s r Ac er-i hef di-a to aH o.)e itha mec

.2. Variations of this basic medium were used to deine the optimal conditions for growth and enzyme s

hesis. Variations concerned the C-source, N-source anucers. Medium III, the basal medium for the preparaf resting cells and fermentation contained per liter 10lycerol, 15.0 g yeast extract, 3.0 g NaCl, 2.0 g KH2PO4,.1 g FeSO4·7H2O, 0.1 g CaCl2·2H2O, and 1.0 gdl-5-(2-

ndolymethyl)hydantoin at pH 7.2.

.4. Biocatalytic assays of resting cells

S. morelensS-5 was cultivated in medium III at 30◦Cor 30 h. Unless otherwise stated, cells were harvestedml of culture broth by centrifugation (10,000×g, 10 min),

wice washed with 0.1 M Tris–HCl buffer (pH 8.0) ae-suspended in 1.0 ml of 0.1 M Tris–HCl buffer (pH 8ontaining 10 mMdl-5-pHPH substrates. The reaction warried out at 40◦C for 1 h after which samples were cerifuged to precipitate the cells and the supernatant wasyzed by HPLC. Other details of the assay are given inable. The activities of the cells inN-carbamoyl-d-pHPG or-pHPG production were expressed in units per milligU/mg) of cell dry mass. One unit was defined as the numf micromoles ofN-carbamoyl-d-pHPG ord-pHPG formeder hour.

.5. Analytical methods

For the quantitative determination of thed-pHPG orN-arbamoyl-d-pHPG formed and thedl-5-pHPH remained ihe bioconversion broth, aliquots were drawn, centrifu

he capillary tube was successively flushed with 0.1 M Naor 2 min, double deionized water for 2 min, followed by ruing buffer (10 mM phosphoric acid containing 40 g/l s

onyl �-cyclodextrin as chiral selective agent, adjusted to.5 with triethanolamine) for 2 min. Samples were disso

n methanol to 0.1 mg/ml and introduced hydrodynamicnto anode end with 0.1 psi for 1 s, then separated. Theemperature was set at 20◦C, and the working voltage wpposite voltage 16 kV. Analyses were detected by UV

ector at 280 nm.

.7. Enzyme preparation

During the purification procedure the temperatureaintained at 4◦C. All columns to which the enzyme solutipplied were equipped on an AKTA FPLC system (Amham Pharmacia Biotech). For the preparation of carbaase, microbial cells, suspended in 50 mM Tris–HCl buH 7.2, 1 mM DTT (buffer A) were disrupted by ultrasoation (400 W, 12 min). The crude enzyme extract was ced by centrifugation (12,000×g, 30 min). The supernataas loaded on a HiPrep16/10Phenyl-hs Column (Ameham Pharmacia Co.) previously equilibrated with buffeontaining 2.0 M NaCl. The enzyme was eluted by lowng the ionic strength of NaCl linearly from 2 to 0 M. Tractions containing enzyme activity were combined andlyzed against buffer A overnight, and then appliediPrep16/10DEAE column (Amersham Pharmacia Cquilibrated with buffer A. After washing the column wlinear gradient of 0–1.0 M NaCl in buffer A, the enzy

ould be eluted at a concentration of 0.1 M NaCl.

522 S. Wu et al. / Enzyme and Microbial Technology 36 (2005) 520–526

2.8. Assay of the partially purified carbamoylase

The cabamoylase activity was assayed according to a mod-ification of the method of Ogawa et al[5]. Unless other-wise stated, the reaction mixtures contained 50 mM sodiumphosphate pH 7.0, 10 mMN-carbamoyl-d-pHPG and the en-zyme in a total volume of 0.4 ml. The reaction proceeded at40◦C for 10–20 min and was stopped with 0.4 ml ethanol.The amount ofd-pHPG formed was determined as describedabove. One unit of the enzyme was defined as the amount ofenzyme that catalyzes the formation ofd-pHPG at the rate of1�mol/min under the conditions of assay.

3. Results

3.1. Isolation and identification of microorganismsproducingd-pHPG

Ten strains, which could utilizeN-carbamoyl-d-pHPGas sole carbon source, were isolated from soil by enrich-ment culture technique. Strain S-5, showing the highestd-carbamoylase activity among these isolates, was selected forfurther characterization. It was a Gram-negative, motile (po-lar flagella), non-spore-forming, obligatory aerobic rod, andc ◦ ◦ alR (ac-c ntitywl icalc tifieda

3

a sed:2 .1 MTo ion

mixture had been incubated at 40◦C for 24 h, the reactionproduct was isolated by preparative HPLC. The colorlesscrystals obtained showed the same patterns of1H NMR andIR spectra as those of authenticN-carbamoyl-d-pHPG ord-pHPG. The absolute configuration ofN-carbamoyl-pHPGand pHPG produced was showed to be thed-enantiomerwhen investigated by CE.Fig. 1 showed the chiral chro-matography of a racemic pHPG,d-pHPG standard and thepHPG produced in the reaction. The pHPG produced showedthe same retention time (15.0 min) as the standardd-pHPG.This indicated the absolute configuration of the productwas ad-enantiomer. The same results were also observedwhen the product ofN-carbamoyl-pHPG was identified.TheN-carbamoyl-pHPG produced exhibited the same reten-tion time (24.9 min) as the standardN-carbamoyl-d-pHPG.The enantiomeric excesses of theN-carbamoyl-pHPG andpHPG produced were found to be >99% ofd-form. Fromthese results, the products of enzymatic hydrolysis ofdl-5-pHPH byS. morelensS-5 were confirmed to be thed-forms of N-carbamoyl-pHPG and pHPG, so hydantoinaseand carbamoylase expressed inS. morelensS-5 wered-stereospecific.

3.3. Effect of different carbon sources

c deda ningc izedi se,s bio-c ofg

3

of1 nu-t inedN ells

ndard

ould grow at 37C but not over 41C. The 16S ribosomNA gene of the stain S-5 was cloned and sequencedession number: AY559079), which had the highest ideith the 16S rRNA sequence ofS. morelense(99% simi-

arity). According to these results and relevant physiologharacteristics (data not showed), the strain S-5 was idensS. morelense.

.2. Isolation and identification of the reaction products

To investigate the configuration of theN-carbamoyl-pHPGnd pHPG produced, the following procedure was u00 mg washed cells were re-suspended in 20 ml of 0ris–HCl buffer (pH 8.0) containing 20 mg ofdl-5-pHPHr N-carbamoyl-dl-pHPG as substrate. After the react

Fig. 1. Chiral chromatography of racemicdl-pHPG (A),d-pHPG sta

To the basal medium II containing yeast extract andN-arbamoyl-d-pHPG, different C-carbon sources were adt a final concentration of 10.0 g/l. The results concerell yield and enzyme activities after 30 h were summarn Table 1. The better growth was observed with glucoucrose and glycerol, but the highest specific activity foronversion ofdl-5-pHPH was obtained by the additionlycerol as carbon source.

.4. Effect of different nitrogen sources

Different nitrogen sources at a final concentration0.0 g/l for organic nutrients and 5.0 g/l for inorganic

rients were added to the basal medium II that conta-carbamoyl-d-pHPG and glucose. Their effects on c

(B),d-pHPG production catalyzed bySinorhizobium morelensS-5 (C).

S. Wu et al. / Enzyme and Microbial Technology 36 (2005) 520–526 523

Table 1Effect of carbon sources on cell growth and formation of enzyme activity

Carbon sources Cell dry mass (CDM)(mg/ml)

d-HPG produced(U/mg cells)

N-Carbamoyl-d-pHPG produced(U/mg cells)

dl-5-pHPH converteda

(U/mg cells)

None 0.56 0.038 0.022 0.060Glucose 0.85 0.056 0.008 0.064Sucrose 1.00 0.029 0.014 0.043Lactose 0.52 0.063 0.031 0.094Sorbose 0.48 0.058 0.023 0.081Soluble starch 0.54 0.056 0.013 0.069Glycerol 0.69 0.084 0.017 0.101Dextran 0.36 0.050 0.017 0.067

a Calculated asN-carbamoyl-d-pHPG plusd-pHPG produced in the standard resting cell assay.

growth and enzyme synthesis were summarized inTable 2.The addition of inorganic nutrients had a negative effect oncell growth and enzyme synthesis, moreover, lessened the ra-tio of d-pHPG toN-carbamoyl-d-pHPG. Although the bestgrowth was observed with soyal peptone, other organic nu-trients including soyal peptone did not show any better ad-vantage than yeast extract for improvement ofdl-5-pHPHhydrolyzing activity (including total activity and specific ac-tivity), so yeast extract was chosen as N-source for furtherinvestigation. Fifteen grams per liter yeast extract was foundto be the optimal concentration for microbial growth and en-zyme synthesis (data not showed).

3.5. Effect of hydantoin derivatives on formation ofenzyme activity

Since hydantoinase and carbamoylase were generallyknown to be induced[3,4], the possible induction effectsof severaldl-5-substituted hydantoins were investigated.The medium containing the indicated hydantoin derivatives(0.1%) in place ofN-carbamoyl-d-pHPG in medium II wasused. As showed inTable 3, the activities of hydantoinaseand carbamoylase were observed regardless of the presenceof a hydantoin derivative during cultivation. But most ofthe hydantoin derivatives could enhance the activities of en-z -s ymesw ass de-

tected hydantoin derivatives, the inducing effect ofdl-5-(2-indolymethyl)hydantoin was highest, which led to 19-foldincrease ofdl-5-pHPH conversion rate, compared to addi-tion of no inducers.

3.6. Hydantoin-hydrolyzing activity during growth

In order to determine at which stage of growth relative en-zyme activity was highest in medium III, the changes indl-5-pHPH-hydrolyzing activity during a batch ofS.morelensS-5were studied. The batch cultivation was performed in a 5-ljar fermentor with 4 l medium at pH 7.2 and 30◦C (Fig. 2).The dl-5-pHPH-hydrolyzing activity of cells increased inparallel with cell growth, and the maximum activity was ob-tained at 30 h, when the culture entered the end of the latelogarithmic growth phase. Upon prolonged incubation, theconversion rate ofdl-5-pHPH decreased distinctly.

3.7. Effect of temperature and pH ond-pHPGproduction by the resting cells of Sinorhizobiummorelens S-5

To optimize the bioconversion, the effects of temperatureand pH optima for thed-pHPG production by the resting cellsof S. morelensS-5 were investigated. As showed inFig. 3Aad n-ci .

TE ctivity

N ced

YFSBT(N

tandard

ymes, which lead to improvement ofdl-5-pHPH converion rate. This indicated that the basal expression of enzas existed inS. morelensS-5, but enzyme expression wtill regulated maybe at transcriptional level. Among the

able 2ffect of nitrogen sources on strain growth and formation of enzyme a

itrogen sources Cell dry mass (CDM)(mg/ml)

d-HPG produ(U/mg cells)

east extract 0.67 0.040ish peptone 0.67 0.040oyal peptone 1.05 0.023eef extract 0.56 0.036ryptone 0.87 0.014NH4)2SO4 0.07 0.014H4NO3 0.04 0.016a Calculated asN-carbamoyl-d-pHPG plusd-pHPG produced in the s

nd B, the optimum temperature and pH ford-pHPG pro-uction were 45◦C and 8.2, respectively. Along with pH irease, hydrolysis ofdl-5-pHPH intoN-carbamoyl-d-pHPGncreased, while the activity ofd-carbamoylase decreased

N-Carbamoyl-d-pHPG produced(U/mg cells)

dl-5-pHPH converteda

(U/mg cells)

0.023 0.0630.016 0.0560.019 0.0420.011 0.0470.012 0.026

0.028 0.0420.020 0.036

resting cell assay.

524 S. Wu et al. / Enzyme and Microbial Technology 36 (2005) 520–526

Table 3Effect of hydantoin derivatives on formation of enzyme activity

Compounds (0.1%) d-HPG produced (U/mg cells) N-Carbamoyl-d-pHPG produced(U/mg cells)

dl-5-pHPH converteda

(U/mg cells)

None 0.062 0.022 0.084Hydantoin 0.274 0.103 0.377dl-Hydantoin-5-acetic acid 0.060 0.031 0.091dl-5-Benylhydantoin 1.277 0.009 1.286dl-5-p-Hydroxyphenylhydantoin 0.327 0.058 0.385dl-5-Phenylhydantoin 0.579 0.056 0.635dl-5-Methylhydantoin 0.188 0.028 0.216dl-5-Methylthioethylhydantoin 0.819 0.071 0.890dl-5-Hydroxymethylhydantoin 0.212 0.013 0.225dl-5-(1-Methylpropyl)hydantion 0.068 0.017 0.085dl-5-Phenylpropylhydantoin 0.807 0.019 0.826dl-5-Isobutylhydantoin 1.101 0.027 1.128dl-5-(2-Indolymethyl)hydantoin 1.579 0.012 1.591

a Calculated asN-carbamoyl-d-pHPG plusd-pHPG produced in the standard resting cell assay.

3.8. Partial physical properties of the partially purifiedd-carbamoylase

N-carbamoyl-d-pHPG hydrolyzing activities measured asa function of temperature from 20 to 80◦C showed that theactivity was highest at 60◦C (Fig. 4A). Upon incubation fordifferent times (from 10 to 60 min) at varying temperatures(from 50 to 60◦C), the enzyme was found to remain 100%activity for over 40 min at 50◦C, and remain 95% activity for20 min at the optimal temperature of 60◦C (Fig. 4B). This in-dicated that the enzyme was thermostable. The enzyme main-tained maximumN-carbamoyl-d-pHPG hydrolyzing activityat pH 7.0 in 100 mM phosphate buffer (Fig. 5).

4. Discussion

Enzymatic production of d-amino acids from 5-substituted hydantoins is catalyzed by two enzymes namely,d-hydantoinase andd-carbamoylase. The cells ofAgrobac-terium[2,4,6], Pseudomonas[7], Arthrobacter[3] andBlas-tobacter [5] showed the two enzyme activities. However,owning to the substrate-specificity and relative activity, thereare only a few strains had the advantage to produce directly

Fi wasdd

of d-pHPG fromdl-5-pHPH. A novel bacterium strain ableto produce directly and enantioselectivelyd-pHPG fromdl-5-pHPH has now been isolated and identified asS. more-lensS-5. Improvement of thed-pHPG production by restingcells could be obtained through optimization of the growthmedium composition. The activities of hydantoinase and car-bamoylase were observed regardless of the presence of ahydantoin derivative during cultivation. However, the ad-dition of dl-5-(2-indolymethyl)hydantoin could lead to 19-fold increase ofdl-5-pHPH conversion rate. The optimum

Fw u-bated under standard conditions at the temperature and pH indicated in thefigure: pH 6.5–7.0, 0.1 M phosphate buffer; pH 7.0–9.0, 0.1 M Tris–HClbuffer: (�) d-pHPG, (�)N-carbamoyl-d-pHPG, (�) total conversion ofdl-5-pHPH.

ig. 2. dl-5-pHPH-hydrolyzing activity ofSinorhizobiummorelensS-5 dur-ng growth in medium III in a 5-l jar fermentor. The course of growthetermined by measuring the cells dry weight. (©) Cells dry weight, (�)-pHPG, (�) N-carbamoyl-d-pHPG, (�) total conversion ofdl-5-pHPH.

ig. 3. Effect of temperature (A) and pH (B) on the production ofd-pHPGith resting cells ofSinorhizobium morelensS-5. Resting cells were inc

S. Wu et al. / Enzyme and Microbial Technology 36 (2005) 520–526 525

Fig. 4. Effect of temperature on the activity (A) and stability (B) of the par-tially purified carbamoylase fromSinorhizobium morelensS-5. (A) Assayswere performed at various temperatures under the enzyme assay conditions.The relative activity is expressed as the percentage of the maximum activityattained under the experimental conditions. (B) The remaining activity wasassayed under the enzyme assay conditions after the enzyme (10 mU) hadbeen placed at indicated temperature for indicated times in 0.2 ml of 50 mMphosphate buffer, pH 7.0. The relative residual activity is expressed as thepercentage of the maximum residual activity attained under the experimentalconditions.

temperature and pH on thed-pHPG production by the restingcells was 45◦C and 8.2, respectively. But under acidic con-dition (at pH 6.5), a complete bioconversion of the racemicdl-5-pHPH intod-pHPG was also observed after increasingreaction times. Because of no chemical racemization occur-

Fig. 5. Effect of pH on the activity of the partially purified carbamoylasefrom Sinorhizobium morelensS-5. The enzyme activity was assayed underthe enzyme assay conditions except that the following. 0.1 M buffers wereused: citric acid/sodium citrate buffer pH 3.8–6.2 (©), sodium phosphatepH 5.7–8.0 (�), Tris–HCl pH 7.1–8.7 (�) and glycin–NaOH pH 8.6–9.0(�).

rence at this pH, these findings indicated that a 5-substitutedhydantoin racemase existed inS. morelensS-5 together withd-hyantoinase andd-carbamoylase. Similar results were alsoobserved inAgrobacteriumsp. IP-I 671[4]. In fact, hydan-toin racemase has been isolated, cloned and expressed fromAgrobacteriumsp. recently[10,11].

The problem of different optimum pH of both enzymes,encountered in a one-step bioconversion process, has beenpointed out by Moller et al.[3] and Runser et al.[4]. Thisproblem also occurred inS. morelensS-5.Fig. 3B showedthe effect of pH on the hydrolysis ofdl-5-pHPH by rest-ing cells. At higher pH (e.g., at pH 9.0), it was favorableto the production ofN-carbamoyl-d-pHPG and adverse toconverting theN-carbamoyl-d-pHPG produced tod-pHPG.This phenomenon may be basically caused by the inhibitionof ammonium on carbamoylase. Runser et al.[4] and Parket al.[12] reported thatN-carbamoylase fromAgrobacteriumsp. was inhibited by ammonium, and the inhibition is moreserious at alkaline conditions than at neutral or acidic pH. Ourstudies showed even 60% enzyme activity of carbamoylase ofS. morelensS-5 had been inhibited when 50 mM ammoniumions were added to the reaction buffer at pH 7.0. In orderto solve the problem, it may be advantageous to convertdl-5-pHPH to produced-pHPG in two separate reaction steps.In the first step, immobilized hydantoinases or cells in thefi -d ingN on-d ondc hy-dp talyzet thet -t rsionp

rewPmA if-f edcaS lee acti-c

R

andric

r-g

rst column hydrolyzedl-5-pHPH to produceN-carbamoyl-pHPG under alkaline conditions. The solution includ-carbamoyl-d-pHPG produced is adjusted to neutral citions by the addition of acid, and applied to the secolumn with immobilized carbamoylases or cells, whichrolyze carbamoyl ofN-carbamoyl-d-pHPG and produced-HPG. Because the hydantoinase and carbamoylase ca

he reactions under the respective optimal conditions inwo-step reaction system, the efficiency ofd-pHPG producion should be higher than that in a one-step bioconverocess.

The optimal temperature ofd-carbamoylase varied moidely. The optima ranged from 40 to 70◦C, showed inseudomonasAJ-11220[13],Agrobacteriumsp.[2,14],Co-amonassp. E222c[15] andBlastobactersp. A17p-4[5].mong these microorganisms,d-carbamoylase exhibited d

erent thermostability. In our studies, the partially purifiarbamoylase exhibited a temperature optimum at 60◦C, andll activity remained after the incubation at 50◦C for 40 min.o thed-carbamoylase ofS.morelensS-5 was a thermostabnzyme, which made it have great potential for use in pral process.

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